The proposed studies address very basic questions about the postnatal growth of phrenic motoneurons and diaphragm muscle (DIAm) fibers. Such growth of phrenic motoneurons and DIAm fibers is necessary to meet the increasing functional diversity of the DIAm. The following issues are a natural extension of the overall goal of determining the role of nerve-muscle interactions on muscle growth. By enhancing vesicular release, BDNF/TrkB signaling could affect release of other nerve-derived trophic factors (e.g., neuregulin, NRG), thereby indirectly promoting postnatal DIAm fiber growth. Therefore, in this revision application we will examine the hypothesis that BDNF/TrkB signaling enhances vesicular release and neuromuscular transmission and, thus, indirectly promotes postnatal growth of DIAm fibers. To address this hypothesis, we propose two new specific aims: Specific Aim 1. To examine the role of BDNF/TrkB signaling on vesicular release and neuromuscular transmission during early postnatal development of the DIAm. We will use two complementary approaches:1) state of the art confocal imaging techniques of synaptic vesicle cycling (FM4-64 fluorescence uptake), and 2) susceptibility to neuromuscular transmission failure during repetitive stimulation of the DIAm. Specific Aim 2. To determine the effect of inhibiting BDNF/TrkB signaling on postnatal growth of DIAm fibers. We will use a recently developed mouse model expressing knockin alleles (TrkBF616A) that permits rapid, reversible and selective inhibition of TrkB kinase activity by membrane-permeable small-molecule protein phosphatase PP1 derivatives (e.g., 1NMPP1). PUBLIC HEALTH RELEVANCE: The diaphragm is the most important inspiratory muscle in mammals;it must provide adequate force to sustain ventilation from birth onwards. It is unique compared to limb muscles, where movement behaviors emerge more gradually after birth. The proposed studies will provide a better understanding of the normal maturation of the diaphragm muscle and the motor neurons that innervate it. In addition, factors that regulate the normal growth and development of the diaphragm will be systematically explored.